JP3649338B2 - Purified Myserioftra laccase and nucleic acid encoding it - Google Patents

Abstract

The present invention relates to isolated nucleic acid constructs containing a sequence encoding a Myceliophthora laccase, and the laccase proteins encoded thereby.

Description

2.過剰の銅の有無での発現
アスペルギルス・オリザ形質転換体HowB104−pRaMB5.30（約10⁹胞子/ml）の胞子懸濁物1mlのアリコートを無菌的に100mlの無菌振盪フラスコ培地（マルトース50g/1;MgSO₄・7H₂O 2g/1;KH₂PO₄ 10g/1;K₂SO₄ 2g/1;GaCl₂・2H₂O 0.5g/1;クエン酸2g/1;酵母抽出物10g/1;微量金属〔ZnSO₄・7H₂O 14.3g/1;CuSO₄・5H₂O 2.5g/1;NiCl₂・6H₂O 0.5g/1;FeSO₄・7H₂O 13.8g/1;MnSO₄・H₂O 8.5g/1;クエン酸3.0g/1〕、0.5ml/1;尿素2g/1;水道水でメスアップし、オートクレーブにかける前にpH6.0に調整）を含む500mlの振盪フラスコに入れ、そして37℃においてロータリーシェーカー上で200rpmにて18時間インキュベーションする。この培養物50mlを無菌的に1.8リットルの発酵培地（MgSO₄・7H₂O 2g/1;KH₂PO₄ 2g/1;クエン酸 4g/1;K₂SO₄ 3g/1;CaCl₂・2H₂O 2g/1;微量金属0.5ml/1;プルロニック発泡抑制剤1ml/1）を含む３リットルの発酵槽に移す。この発酵槽の温度は発酵槽ジャケットを通じる冷却水の循環により34℃に保つ。無菌エアーを1.8リットル／分（1v/v/m）の率で発酵槽に散布する。撹拌速度は培養物中の溶解酸素レベルを20％より高く保つのに必要とされるほぼ最低レベルである。600〜1300rpmに維持する。無菌添加物（Nutriose 725〔マルトースシロップ〕225g/1;尿素30g/1;酵母抽出物15g/1;プルロニック発泡抑制剤1.5ml/1;蒸留水でメスアップし、そしてオートクレーブにかける）をペリスターポンプを利用して発酵槽に加える。発酵の際の供給速度は以下の通りとする：接種前は当初30gの添加物;0〜24h 2g/1h;24〜48h 4g/1h;48h−終了 6g/1h。
銅を水又は適当なバッファー中で400×のストックとして調製し、無菌濾過し、そして無菌的にタンクに0.5mMの最終レベルとなるように加える。上記の発酵をタンク培地への銅添加物の添加抜きでも行う。酵素活性の決定のためのサンプルを抜き取り、そしてMiraclothで濾過して菌糸体を除去する。これらのサンプルを上記のLACUアッセイによりラッカーゼ活性についてアッセイする。ラッカーゼ活性は発酵中に連続的に上昇することが認められ、過剰の銅を含む発酵においては180時間後に約45LACU/mlの値が達成される。22LACU/mgの比活性において、これは2g/1の発現組換ラッカーゼに相当する。他方、銅添加物抜きの発酵において達成される最大ラッカーゼ活性は170時間後に約10LACU/mlであり、添加銅の存在下で見い出せる値の約25％であった。
III.マイセリオフトラ・ラッカーゼの精製及び特性決定
A.材料及び方法
1.材料
バッファー及び基質として用いる化学試薬は最低でも試薬級の商品とする。エンド/N−グリコシダーゼＦ及びパイログルタミン酸アミノペプチダーゼをBoehringer Mannheimより購入した。クロマトグラフィーはPharmacia FPLC又は慣用の低圧システムのいづれかで行う。吸光アッセイは光度計（Shimadzu PC160）又はマイクロプレートリーダー（Molecular Devices）のいづれかで行う。Britton ＆ Robinson（Ｂ＆Ｒ）バッファーをQuelle,Biochemisches Taschenbuch,H.M.Raven,II.Teil,S.93u,102,1964に記載のプロトコールに従って調製する。
2.酵素活性
ラッカーゼ活性はシリンガルダジン酸化により、30℃にて１−cm石英キュベットの中で決定する。60μ１のシリンガルダジンストック溶液（50％のエタノール中0.28mM）及び20μ１のサンプルを0.8mlの予備加熱したバッファー溶液と混合する。酸化は５分間にわたり530nmでモニターする。活性は１分間当りに酸化された基質のμmoleとして表示する。様々なpHのＢ＆Ｒバッファーを使用する。活性単位はここでは「SOU」と称する。上述の通りLACUと称する活性を決定するために25mMの酢酸ナトリウム、40μＭのCuSO₄,pH5.5のバッファーも使用する。2,2'−アジノビス（３−エチルベンゾチアゾリン−６−スルホン酸）（ABTS）酸化アッセイは0.4mMのABTS,B＆Ｒバッファー、pH4.1を用い、室温においてA₄₀₅をモニターすることにより行う。ABTSオキシダーゼ活性上層（オーバーレー）アッセイは、冷却したABTS−アガロース（0.05gのABTS,1gのアガロース、50mlのH₂O、アガロースを溶かすために加熱）を自然IEFゲルの上に注ぎ、そして室温でインキュベートすることにより行う。ラッカーゼ（ｒ−MtL）の熱安定性分析は、Ｂ＆ＲバッファーpH6の中で様々な温度において予備インキュベーションした3SOU活性を有するサンプルを用いて行う。サンプルは同じバッファーに400倍に希釈してから室温でアッセイする。
3.発酵培養液からの精製
3.7リットルのチーズクロス濾過した培養液（pH7.6,16mS）をWhatman＃２濾紙で濾過する。その培養液をS1Y100膜（MWCO:100）の付いたSpiral Concentrator（Amicon）で3700mlから200mlへと濃縮する。その濃縮液を水に希釈することにより0.75mSに調整し、そしてS1Y100で170mlに再濃縮する。洗浄且つ濃縮した培養液は濃緑色を帯びた色調を有する。
その培養液を−20℃で一夜凍結し、翌日融解し、そして10mMのトリス、pH7.5,0.7mS（バッファーＡ）で予備平衡化しておいたＱ−sepharose XK26カラム（120ml）に載せる。青色のラッカーゼバンドは添加中にカラムをゆっくり下降する。青色画分の１のグループは添加及びバッファーＡによる洗浄の後はカラムを通り抜ける。第２グループはバッファーＢ（バッファーＡと2MのNaCl）による線形勾配の際に溶出する。ラッカーゼ活性のない一部の茶色の物質は1MのNaOHによりその後溶出する。SDS−PAGE分析はこの調製が純粋なラッカーゼをもたらすことを示す。
4.アミノ酸含有量、グリコシル化の程度及びＮ−末端配 列の分析
Ｎ−末端配列決定をABI 476Aシーケンサーで行う。全アミノ酸分析（それからｒ−MtLの励起係数を決定）をHP Amino Quant装置で実施する。脱グリコシル化はエンド/N−クルコシダーゼＦを用いて製造業者の仕様書に従って行い、そして炭水化物含有量はSDS−PAGEにより決定される移動度の相違により評価する。パイログルタミン酸アミノペプチダーゼによるＮ−末端の脱ブロッキングは製造業者の仕様書に従って実施する。約80μｇのｒ−MtLを４μｇのペプチダーゼにより、1Mの尿素又は0.1MのグアニジンHClの存在下又は非存在下で処理し、次いで配列決定のためにPVDF膜にブロッティングする。約20pmolの脱ブロッキングされたタンパク質が得られ、そして配列決定する。
SDS−PAGE及び自然IFF分析はNovexセル又はMini Protean II及びモデルIII Mini IEFセル（Bio−Rad）のいづれかで行う。ゲル濾過分析はSephacryl S−300（Pharmacia）で行い、それより自然MWをカラムを較正するためのブルーデキストラン（2000kdal）、牛IgG（158kdal）、牛血清アルブミン（66kdal）、オバルブミン（45kdal）及び馬心臓ミオグロビン（17kdal）を用いることにより推定する。
B.結果及び考察
1.発酵培養液からのｒ−MtLの精製及び特性決定
3.71の発酵培養液から、約２〜3gのｒ−MtLが単離される。100kdalのMWCOを有する膜を用いる最初の濃縮は有意な量の茶色物質及び少量の夾雑タンパク質を除去した。10mMのトリス、pH7.5で平衡化しておいたＱ−Sepharoseマトリックスに対するｒ−MtLの低親和力はその他のより酸性、且つより強く結合した不純物からのその分離を助長する。SDS−PAGEにより示される通り、この調製はピークのまわりに位置している最も活性な画分に関する本質的に純粋ラッカーゼをもたらした。その他の活性の弱い画分はより浅い勾配によるMon−Ｑ又はゲル濾過カラム、例えばＳ−300のいづれかで更に精製でき、それより夾雑物は小さめのMWに基づき分離される。総合的に18倍の精製度及び67％の回収率が達せられる。以下に記載の通り、Ｑ−Sepharoseクロマトグラフィーでのｒ−MtLの２本の溶出バンドの存在はおそらくは示差的なグリコシル化に基づく。
精製したｒ−MtLはＳ−300ゲル濾過で100〜140kdalのMW、そしてSDS−PAGEで85kdalのMWを示す。脱グリコシル化後のSDS−PAGEでのｒ−MtLの移動度の上昇は炭水化物がその全質量の14％を占めることを示す。自然IEFはABTSオーバーレーアッセイにおいて活性なpI〜4.2の主要バンドを示した。
脱塩した溶液又はPVDF膜のいづれかのサンプル由来の精製ｒ−MtLのＮ−末端の直接配列決定は不成功に終わった。しかしながら、パイログルタミン酸アミノペプチダーゼによるｒ−MtLの処理は脱ブロッキングされたＮ−末端を有するタンパク質をもたらした。これは、ｒ−MtLの成熟化の際のプロペプチドのプロセシング、即ち、リゾクトニア・ソラニ（Rhizoctonia solani）の如きその他のラッカーゼにおいては認められないが、N.クラッサ・ラッカーゼのそれに類似する後翻訳現象を示唆する。考えられるスキームを以下に概略する。

青色のｒ−MtLのスペクトルは276及び589nmにて最大吸収を有していた。
ラッカーゼの活性を基質としてシリンガルダジン及びABTSのいづれかを用いて試験する。Abs₂₇₆当り又はmg当りとして表示して、ラッカーゼはpH6.5において20又はSOUに関する45単位の値それぞれを有した。LACUアッセイはAbs₂₇₆当り又はmg当り10又は22単位の値をもたらした。
ｒ−MtL活性のpHプロフィールは野生型のそれとかなり近く、6.5の至適pHを有する。ｒ−MtLについて観察される20分の予備インキュベーション後に保持している完全活性にとっての上記温度値は約60℃である。精製ｒ−MtLはＱ−Sepharose溶出バッファーの中で−20℃で凍結して５週間保存しても活性を失わないことを示した。
Ｑ−Sepharoseで単離した発酵培養液から得られるｒ−MtLの２通りの形態を比較したとき、SDS−PAGE、自然PAGE、自然IEF,S−300ゲル濾過、UV可視スペクトル、シリンガルダジン及びABTSに対する比活性、並びに脱ブロッキングしたＮ−末端配列決定尺度の観点において有意な差はなかった。同様に、Ｑ−Sepaharoseでの種々の溶出パターンは数種の異なるグリコシル化に由来する。
IV.毛髪の染色におけるマイセリオフトラ・ラッカーゼ の利用
マイセリオフトラ・ラッカーゼの染色効果を様々な染料前駆体に基づき、そして更にはいく種かの改質剤と対比させた0.1％ｐ−フェニレンジアミンに基づいて試験した。
材料：
染料前駆体：
0.1MのＫ−リン酸バッファー（pH7.0）中の0.1％のｐ−フェニレン−ジアミン
0.1MのＫ−リン酸バッファー（pH7.0）中の0.1％のアミノフェノール
酵素：
組換マイセリオフトラ・サーモフィラ・ラッカーゼ16LACU/ml（最終染色溶液中）。
装置：
Datacolor Textflash 2000（CIE−Lab）
毛髪の色の評価
巻き毛の定性的色調をDatacolor Textflash 2000で、CIE−LabパラメーターＬ^＊（「０」＝黒、そして「100」＝白）とａ^＊（「−」＝緑、そして「＋」＝赤）を利用して決定する。
結果：
染色効果
ヨーロッパ人の金髪の巻き毛（1g）を酸化性毛髪染色についてマイセリオフトラ・サーモフィラ・ラッカーゼを試験するために用いる。染料前駆体としてｐ−フェニレンジアミン及びｏ−アミノフェノールを使用する。
毛髪染色
4mlの染料前駆体溶液をWhirleyミキサーで1mlのラッカーゼと混合し、巻き毛に塗布し、そして30℃で60分保つ。その巻き毛を水道水で約３すすぎ、２本の指の間で押え、くしを通し、そして風乾する。
染色効果試験の結果を以下の表１及び２に示す。

試験の結果
表１及び２から、マイセリオフトラ・サーモフィラ・ラッカーゼが毛髪の酸化的染色のために利用できることがわかる。
生物材料の寄託
以下の生物材料をブダペスト条約のもとで、1994年５月25日にAgricultural Research Service Patent Culture Collection,Nothern Regional Research Center,1815 University Street,Peoria,Illinois,61604に寄託し、そして以下の受託番号が与えられている。
寄託物 受託番号
pRaMB5含有のE.コリJM101 NRRL B−21261
配列表
（１）一般情報：
（ｉ）出願人：
（Ａ）名称:Novo Nordisk Biotech,Inc.
（Ｂ）通り:1445 Drew Avenue
（Ｃ）市:Davis,California
（Ｄ）国:United States of America
（Ｅ）郵便番号:95616−4880
（Ｆ）電話番号：（916）757−8100
（Ｇ）ファックス番号：（916）758−0317
（ｉ）出願人：
（Ａ）名称:Novo Nordisk A/S
（Ｂ）通り:Novo Alle
（Ｃ）市:Bagsv rd
（Ｄ）国:Denmark
（Ｅ）郵便番号:DK−2880
（Ｆ）電話番号：＋45 4444 8888
（Ｇ）ファックス番号：＋45 4449 3256
（ii）発明の名称：精製されたマイセリオフトラ・ラッカーゼ及びそれをコードする核酸
（iii）配列の数:2
（iv）連絡先：
（Ａ）名称:Novo Nordisk of North America,Inc.
（Ｂ）通り:405 Lexington Avenue,Suite 6400
（Ｃ）市及び州:New York,New York
（Ｄ）国:U.S.A.
（Ｅ）郵便番号:10174−6401
（ｖ）コンピューター読取フォーム：
（Ａ）媒体タイプ:Floppy disk
（Ｂ）コンピューター:IBM PC compatible
（Ｃ）作動システム:PC−DOS/MS−DOS
（Ｄ）ソフトウェア:PatentIn Release＃1.0,Version＃1.25（EPO）
（vi）現出願人データー：
（Ａ）出願番号：認定前
（Ｂ）出願日:1995年５月31日
（Ｃ）分類：
（vii）先の出願のデーター：
（Ａ）出願番号:US 08/253,781
（Ｂ）出願日:1994年６月３日
（viii）代理人／代理店情報：
（Ａ）名称:Lowney,Karen A.
（Ｂ）登録番号:31,274
（Ｃ）参照／事件番号:4184.204−WO
（ix）通信情報：
（Ａ）電話番号:212 867 0123
（Ｂ）ファックス番号:212 867 0298
（２）SEQ ID NO:1についての情報：
（ｉ）配列の特徴：
（Ａ）長さ:3187塩基対
（Ｂ）タイプ：核酸
（Ｃ）鎖の数：二本鎖
（Ｄ）トポロジー：直鎖
（ii）分子のタイプ:DNA（ゲノム）
（vi）起源：
（Ａ）生物：マイセリオフトラ・サーモフィラ
（ix）特徴：
（Ａ）名称／キー：イントロン
（Ｂ）位置:833...917
（ix）特徴：
（Ａ）名称／キー：イントロン
（Ｂ）位置:996...1077
（ix）特徴：
（Ａ）名称／キー：イントロン
（Ｂ）位置:1090...1188
（ix）特徴：
（Ａ）名称／キー：イントロン
（Ｂ）位置:1261...1332
（ix）特徴：
（Ａ）名称／キー：イントロン
（Ｂ）位置:2305...2451
（ix）特徴：
（Ａ）名称／キー：イントロン
（Ｂ）位置:2521...2613
（ix）特徴：
（Ａ）名称／キー:CDS
（Ｂ）位置：連絡（587..832,918..995,1078..1089,1189..1260,1333..2304,2452..2520,2614..3024）
（xi）配列の詳細:SEQ ID NO:1:

（２）SEQ ID NO:2についての情報：
（ｉ）配列の特徴：
（Ａ）長さ:620アミノ酸
（Ｂ）タイプ：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖
（ii）分子のタイプ：タンパク質
（vi）起源：
（Ａ）生物：マイセリオフトラ・サーモフィラ
（xi）配列の詳細:SEQ ID NO:2:

Field of Invention
The present invention relates to an isolated nucleic acid enzyme fragment encoding a fungal oxidoreductase enzyme and a purified enzyme produced thereby. More particularly, the present invention relates to a nucleic acid fragment encoding a phenol oxidase, in particular a laccase of the thermophilic Ascomycetes Myceliophthora.
Background of the Invention
Laccase (benzenediol: oxygen oxidoreductase) is a polycopper-containing enzyme that catalyzes the oxidation of phenols. Laccase-mediated oxidation results in the production of an aryloxy group intermediate from a suitable phenolic substrate. The ultimate coupling of the intermediate thus produced provides a combination of dimer, oligomer and polymerization reaction product. Such reactions are essential in the biosynthetic pathway that results in the formation of melanin, alkaloids, toxins, lignin and humic acid. Laccases can be used in a wide variety of fungi such as Ascomycetes, such as Aspergillus, Neurospora, and Podospora, incomplete fungi Botrytis, and basidiomycetes, such as Collybia, Homes. (Fomes), Lentinus, Pleurotus, Trametes, and Rhizoctonia. Laccases exhibit a wide range of substrate specificities, and the various fungal laccases usually differ from each other in their ability to oxidize phenolic substrates. Because of substrate diversity, laccases together find many potential industrial uses. Among them are lignin modification, paper strengthening, inhibition of dye transfer in detergents, phenol polymerization, juice production, phenol resin production and wastewater treatment.
The catalytic ability of laccases made by different fungal species is similar or has different optimum temperatures and pHs, and these may differ depending on the particular substrate. A number of these fungal laccases have been isolated and the genes for some of these have been cloned. For example, Choi et al. (Mol. Plant-Microbe Interactions5: 119-128,1992) describes the molecular characterization and cloning of the gene encoding the laccase of the chestut blight fungus Cryphonectria parasitica. Kojima et al. (J. Biol. Chem.265: 15224-15230,1990; JP2-23885) provides a description of two alleles of the laccase of the white-rot basidiomycete Coriolus hirsutus. Germann and Lerch (Experientia41: 801,1985; PNAS USA83: 8854-8858, 1986) report the cloning and partial sequencing of the Neurospora crassa laccase gene. Saloheimo et al. (J. Gen. Microbiol.137: 1537-1544, 1985; WO92 / 014046) disclose the structural analysis of the laccase gene from the fungus Phlebia radiata.
Attempts to express laccase genes in heterologous fungal systems often result in very low yields (Kojima et al., Supra; Saloheimo et al., Bio / Technol.9: 987-990,1991). For example, heterologous expression of flavia radiata laccase in Trichoderma reesei provides only 20 mg of active enzyme per liter (Saloheimo, 1991, supra). Laccase has great commercial potential, but the ability to express enzymes in large quantities is important for its commercial use. At present, there are no laccases that are expressed at high levels in commercially available hosts such as Aspergillus. That is, there is a need for the presence of laccase that can be produced in commercially useful quantities (ie, grams / liter or more). The present invention meets this need.
Summary of the Invention
The present invention relates to a DNA construct comprising a nucleic acid sequence encoding Myserioftra laccase. The invention further relates to an isolated laccase encoded by a nucleic acid sequence. Preferably, the laccase is substantially pure. “Substantially pure” means that the laccase is essentially free of other non-laccase proteins (ie, ≧ 90%).
To facilitate the production of novel laccases, the present invention further provides vectors and host cells comprising the claimed nucleic acid sequences, which are useful in the recombinant production of laccases. This sequence is operably linked to transcriptional and translational signals that can direct the expression of the laccase protein in the selected host cell. Preferred host cells are fungal cells, most preferably those of the genus Aspergillus. Recombinant production of the laccase of the present invention involves culturing host cells transformed or transfected with the construct of the present invention or progeny thereof under conditions suitable for expression of the laccase protein, and laccase protein from the culture. This is achieved by recovering.
The laccases of the present invention are useful in a number of industrial processes that require the oxidation of phenols. These processes include lignin treatment, juice production, phenol polymerization and phenol resin production.
[Brief description of the drawings]
FIG. 1 shows a restriction map of the 7.5 EcoR I fragment in pRaMB1. The region that hybridizes with the N. crassa laccase gene probe is shaded.
FIG. 2 shows the nucleotides (SEQ ID NO: 1) and amino acids (SEQ ID NO: 2) of M. thermophila laccase. The lower case lower letter in the nucleotide sequence indicates the position of the intron. Putative TATA and CAAT sequences in the promoter region are shown in bold and underlined. The common lariat construction (PuCTPuAC) in the intron is underlined.
FIG. 3 shows the construction of plasmid pRaMB5.
Detailed Description of the Invention
Myseri offora thermophila was first discussed by Apinis (Nova Hedwigia5: 57-78,1963), and a thermophilic ascomycota class called Sporotrichum thermophile. Subsequent taxonomic correction caused the organism to belong to the genus Chrysosporium (Von Klopotek, A. Arch. Microbiol.98: 365-369,1974), and later belonged to Myseriophora (Van Oorschot, Persoonia)9: 401-408,1977). Several organisms known by other names were also found to belong to this species. They are Sporotricum. S. cellulophilum (US Pat. No. 4,106,989); Thielavia thermophila (Fergus and Sinden, Can. J. Botany 47: 1635-1637, 1968); Chrysosporium fergashi (C. fergussi) and Corynascus thermophilus (Von Klopotek, supra). This species is known as the origin of several different industrially useful enzymes such as cellulase, β-glucosidase and xylanase (see, eg, Oberson et al., Enzyme Microb. Technol.14: 303-312,1992; Merchant et al., Biotechnol.Lett.Ten: 513-516, 1988; Breuil et al., Biotechnol. Lett.8: 673-676, 1986; Gilbert et al., Bioresource Technol.39: 147-154,1992). It has now been determined that Myseri offtra produces a neutral pH laccase and that the gene encoding this laccase can be used to produce conventional host systems such as Aspergillus in large quantities.
To identify the presence of the laccase gene in Myseri offtra, the 5 ′ region (lcc 1) of the Neurospora crassa laccase gene is subject to mild stone-generic conditions in Southern hybridization of total genomic DNA of various fungal species. Used as a probe. An approximately 12 kb laccase specific sequence is detected in Myseri offtra DNA. The N. classa fragment is then used to screen about 20,000 plaques of the M. thermophila genomic DNA library in the λEMBL4 bacteriophage cloning vector. Eight plaques hybridize strongly with this probe. DNA was isolated from three of the eight. Each of these clones contains a 7.5 EcoR I fragment, which also hybridizes to the probe (FIG. 1). One of the fragments was subcloned into pBR322 to create plasmid pRaMB1. Using the lcc 1 probe, the position of the coding region of the clone is determined. The entire M. thermophila coding region was found to contain a 3.2 kb Nhe-I Bgl II segment, which was cloned into pUC119 and sequenced by primer walking.
Once the sequence has been determined, the location of introns and exons within the gene is identified based on the predicted amino acid sequence match to the corresponding N. classa laccase gene product. From this contrast, the M. thermophila gene (lccM) consists of seven exons (246,79,12,70,973,69 and 411 nucleotides) intervened by six introns (85,84,102,72,147 and 93 nucleotides). It becomes clear. The coding region, excluding the intervening sequence, is very GC rich (65.5% G + C) and a 620 amino acid preproenzyme: a mature laccase comprising a 22 amino acid signal peptide, a 25 amino acid propeptide and 573 amino acids. Code. The sequence of the M. thermophila gene and the deduced amino acid sequence are shown in FIG. 2 (SEQ ID NO: 1 and 2).
The laccase gene is then utilized to construct an expression vector for transformation of Aspergillus host cells. This vector pRaMB5 contains the A. oryzae TAKA-amylase promoter and terminator region. The construction of pRaMB5 is outlined in FIG. Aspergillus cells are cotransformed with this expression vector and a plasmid containing a pyrG or amds selection marker. Transformants are selected based on an appropriate selection medium containing ABTS. Laccase-producing colonies show a green circle and can therefore be easily isolated. Selected transformants were grown in shake flasks and the culture medium was tested for laccase activity by the syringaldazine method. Shake flask cultures can produce 0.2 g / 1 or more of laccase and yields in the fermenter of greater than 1-2 g / 1.
In accordance with the present invention, the myseriophora gene encoding laccase can be obtained by the methods described above or by other methods known in the art using the information provided herein. This gene can be expressed in an active form using an expression vector. Useful expression vectors are those that allow stable integration of the vector into the host cell genome or self-replication of the vector in the host cell independent of the host cell genome, and preferably easy selection of transformed host cells. One or more phenotypic markers that enable The expression vector may further comprise a promoter, a ribosome binding site, a translation initiation signal, and optionally a control sequence encoding a repressor gene or various activating genes. In order to allow secretion of the expressed protein, a nucleotide encoding a signal sequence may be inserted in front of the coding sequence of the gene. For expression under the direction of control sequences, the laccase gene utilized in accordance with the present invention is operably linked to the control sequence within the appropriate reading frame. Promoter sequences that can be incorporated into plasmid vectors and can direct transcription of the laccase gene include, but are not limited to, the prokaryotic β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA75: 3727-3731) and the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80: 21-25) is included. For further reference, see `` Useful proteins from recombinant bacteria '' Scientific American, 1980,242: 74-94 and Sambrook et al., "Molecular Cloning" 1989.
The expression vector carrying the DNA construct of the present invention may be any vector that can be easily submitted to recombinant DNA procedures, and the choice of vector will generally depend on the host cell into which it is introduced. Let's go. That is, the vector can be a self-replicating vector, ie, a vector that exists as a foreign chromosome and whose replication is independent of chromosomal replication, such as a plasmid, or extrachromosomal factors, minichromosomes, or artificial chromosomes. On the other hand, when introduced into a host cell, the vector may be one that integrates into the host cell genome and replicates along with its integrated chromosome.
Within the vector, the laccase DNA sequence should be operably linked to a suitable promoter sequence. The promoter can be any DNA sequence that exhibits transcriptional activity in the selected host cell and can be derived from a gene encoding a protein that is either cognate or heterologous to the host cell. Examples of promoters suitable for directing transcription of the DNA constructs of the present invention, particularly promoters in bacterial hosts, include the E. coli lac operon promoter, Streptomyces coelicolor agarase Gene dagA promoter, Bacillus licheniformis α-amylase gene (amyL) promoter, Bacillus stearothermophilus maltogenic amylase gene (amyM) promoter, Bacillus amyloliquefacies ( B. amyloliquefaciens) α-amylase (amyQ) promoter, B. subtilis xylA and xylB gene promoter. A useful promoter in yeast hosts is the eno-1 promoter. Examples of useful promoters for transcription in fungal hosts include A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid-stable α-amylase, A. niger or A. awamori glucoamylase (glaA), Rhizomucor mihei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, Or derived from the gene encoding A. nidulans aselemidase. TAKA-amylase and the glaA promoter are preferred.
The expression vector of the present invention may comprise a suitable transcription terminator and, in eukaryotic cells, may also comprise a polyadenylation sequence operably linked to a DNA sequence encoding the laccase of the present invention. . Transcription and polyadenylation sequences may suitably be derived from the same source as the promoter. The vector may further comprise a DNA sequence that enables the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACY177, pUB110, pE194, pAMB1 and pIJ702.
This vector further comprises a selectable marker, for example a gene whose product complements the defect in the host cell, for example the dal gene from B. subtilis or B. licheniformis, or antibiotic resistance, for example ampicillin, kanamycin, chloramphenicol or tetracycline resistance It may also comprise a gene that confers. Examples of Aspergillus selectable markers include amdS, pyrG, argB, niaD, sC and the marker hygB that confers hygromycin resistance. Preferred for use in Aspergillus host cells are A. nidulans or A. oryzae amdS and pyrG markers. A frequently used mammalian marker is the dihydrofolate reductase (DHFR) gene. Furthermore, selection can be accomplished by co-transformation as described in WO91 / 17243.
In general, expression preferably results in a product that is extracellular. The laccases of the present invention may therefore comprise a pre-region that causes the expression medium to secrete the expressed protein. If desired, this pre-region can be natural to the laccase of the present invention, or it can be replaced by a pre-region or signal sequence, which is conveniently accomplished by replacement of the DNA sequence encoding the pre-region of the reaction. For example, the pre-region is a glucoamylase derived from Aspergillus sp. Or an amylase gene, an amylase gene derived from Bacillus sp., A lipase or proteinase gene derived from Rhizomucor maihei, Saccharomyces cerevisiae or a calf-derived α-factor. It can be derived from a gene. Particularly preferably, when the host is a fungal cell, A. oryzae TAKA amylase, A. niger neutral amylase, maltogenic amylase from Bacillus NCIB 11837, B. stearothermophilus α-amylase or Bacillus licheniformis Subtilisin. Valid signal sequences are the A. oryzae TAKA amylase signal, the Rhizomucor mihei aspartic proteinase signal, and the Rhizomucor mihei lipase signal.
The procedures used to ligate the DNA constructs, promoters, terminators and other factors of the invention, respectively, and insert them into appropriate vectors containing information essential for replication are known to those skilled in the art (e.g., See Sambrook et al., Molecular Cloning, 1989).
A cell of the invention comprising any of the DNA constructs or expression vectors of the invention described above is conveniently used as a host cell in the recombinant production of the enzyme of the invention. This cell can be transformed with the DNA construct of the invention, conveniently by integrating the DNA construct into the host chromosome. This integration is generally considered convenient because the DNA sequence tends to be stably maintained in the cell. Integration of the DNA construct into the host chromosome can be carried out according to conventional methods, for example by homology or heterologous recombination. Alternatively, the cell can be transformed with the expression vector as described above in connection with various types of host cells.
The host cell can be selected from prokaryotic cells, such as bacterial cells. Examples of suitable bacteria are Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, B. lentus, B. brevis, Bacillus stearothermophilus, Bacillus alkalophilus ( B. alkalophilus), Bacillus amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium, Bacillus -B. thuringiensis, or Streptomyces lividans or S. murinus, or Gram-negative bacteria such as E. coli. Bacterial transformation may be performed, for example, by protoplast transformation or by utilizing competent cells in a manner known per se.
Host cells may be eukaryotic, such as mammalian cells, insect cells, plant cells or preferably fungal cells such as yeasts and filamentous fungi. For example, useful mammalian cells include CHO or COS cells. Yeast host cells may be selected from Saccharomyces or Schizosaccharomyces species such as Saccharomyces cerevisiae. Useful fungi can be selected from Aspergillus species, such as Aspergillus oryzae or Aspergillus niger. On the other hand, strains of Fusarium species such as F. oxysporum can be used as host cells. Fungal cells may be transformed by protoplast formation and protoplast transformation in a manner known per se, followed by cell wall regeneration. A suitable procedure for transformation of Aspergillus host cells is described in EP 238,023. A suitable method for transforming Fusarium species is described by Malardier et al., 1989.
Accordingly, the present invention provides a method for producing the recombinant laccase of the present invention, wherein the method comprises culturing the above host cell under conditions that induce the production of the enzyme and recovering the enzyme from the cell and / or culture medium. Comprising. The medium used for culturing the cells may be any conventional medium suitable for growing the subject host cells and obtaining expression of the laccase of the present invention. Appropriate media are available from commercial suppliers or can be prepared according to published recipes (eg, listed in catalogs of the American Type Culture Collection).
In a preferred embodiment, recombinant production of laccase in the culture is achieved in the presence of excess copper. Although the trace metals added to the culture medium contain a small amount of copper, experiments conducted in the context of the present invention show that the addition of copper additives to the medium can increase the yield of active enzyme many times. Preferably, copper is added in a soluble form in the medium, preferably in the form of a soluble copper salt, for example in the form of copper chloride, copper sulfate or copper acetate. The final concentration of copper in the medium should be in the range of 0.2 to 2 mM, preferably in the range of 0.05 to 0.5 mM. This method can be used to increase the yield of any recombinantly produced fungal laccase and other copper-containing enzymes, particularly oxidoreductases.
The resulting enzyme is separated from the medium by conventional procedures such as centrifugation or filtration, the cells are separated from the medium, the protein component of the supernatant or filtrate is precipitated with a salt such as ammonium sulfate, and then various chromatographic procedures such as ions It can be recovered by purification by exchange chromatography, gel filtration chromatography, affinity chromatography or the like. Preferably, the isolated protein is about 90% pure as determined by SDS-PAGE, the purity of which is most important in food, juice or detergent applications.
In particularly preferred embodiments, expression of laccase is achieved in fungal host cells, such as Aspergillus. As described in detail in the Examples below, the laccase gene is ligated into a plasmid containing the Aspergillus origa TAKAα-amylase promoter and the Aspergillus nidulans amdS selectable marker. On the other hand, amdS is on an independent plasmid and is available for co-transformation. One or more plasmids are utilized to transform Aspergillus spp. Host cells such as A. oryzae or A. niger according to the method described by Yeltor et al. (PNAS USA 81: 1470-1474, 1984).
One skilled in the art will appreciate that the present invention is not limited to the use of the nucleic acid fragments disclosed in detail herein, such as that in FIG. It will also be apparent that the present invention encodes the same amino acid sequence as shown in FIG. 1, but encompasses nucleotide sequences that differ from the indicated nucleotide sequence due to the degeneracy of the genetic code. Also, references to FIG. 1 in the specification and claims are understood to encompass the genomic sequences described therein and the corresponding cDNA and RNA sequences, and as used herein “DNA constructs” and It will be understood that the term “nucleic acid sequence” encompasses all variants thereof. “DNA construct” is generally understood to mean either a single-stranded or double-stranded DNA molecule, which is isolated in partial form from a natural gene or is not present in nature. Modified to include segments of DNA that are bound and aligned in
The myserioftra laccase described herein has a very high specific activity for syringaldazin substrates compared to other known ascomycetes or incomplete fungi whose specific activity is discussed. The present invention provides a means by which other ascomycetes and / or incomplete fungal laccases can also be isolated. Identification and isolation of laccase genes from sources other than those specifically exemplified herein can be accomplished by publicly available ascomycetes and incomplete fungal strains using the methods described in this example. sell. In particular, the specific sequences disclosed herein can be used to design primers and / or probes useful in isolating similar laccase genes by standard PCR or Southern hybridization techniques. Accordingly, the present invention encompasses ascomycetes and incomplete fungal laccases having a specific activity of about 30 SOU / mg or more, and preferably about 40 SOU / mg or more. “SOU” is defined as the μmole amount of substrate that is oxidized per minute as measured using syringaldazine as the substrate at the optimum pH.
Furthermore, the present invention encompasses other myseriophora laccases, for example other forms of laccase that may be found in M. thermophila, and other belonging to the definition of Myserioftra according to the definitions by Van Oorschot, 1977, supra. Contains laccase that can be found in fungi. Identification and isolation of laccase genes from sources other than those specifically exemplified herein can be accomplished using publicly available Myserioftra strains by utilizing the methods described in this example. On the other hand, the sequences disclosed herein can be used to design primers and / or probes useful in isolating laccase genes by standard PCR or Southern hybridization techniques. Other names of Myseriofra species include M. hinnulea (Awao et al., Mycotaxon,16: 436-440,1983), M. vellerea (Guarro et al., Mycotaxon,twenty three: 419-427,1985) and M. lutea Costantin. Also encompassed are alias laccases, such as the anamorphs or complete state of Myseriophora species or strains. Myceliotra strains are readily publicly accessible at a number of culture depository institutions. For example, ATCC 48102, 48103, 48104, etc .; CBS 117.65, 131.65, 379.65, etc .; DSM 1799 (M. thermophila), ATCC 52474, CBS 539.82, 540.82 etc. (M. Hinnurea), DSM 62114, DBS 146.50, 147.50, 157.51, etc. (M. lutea), and CBS 478.76, 479.76 and 715.84 (M. Belerea). The invention further encompasses any mutated nucleotide sequence and the protein encoded thereby, which protein is about 80% or more, preferably 85% or more, and most preferably 90-95% or more of the amino acid sequence shown in FIG. And qualitatively retains the laccase activity of the sequences described herein. Useful variants within the above categories include, for example, those with conservative amino acid substitutions, and such substitutions should not significantly affect the activity of the protein. A conservative substitution means that an amino acid of the same class can be replaced by any other member of that class. For example, the nonpolar aliphatic residues Ala, Val, Leu and Ile may be interconverted in the same way as the basic residues Lys and Arg, or the acidic residues Asp and Glu. Similarly, Ser and Thr, like Asn and Gln, are in a conservative substitution relationship with each other. It will be apparent to those skilled in the art that such substitutions can be made outside the region important for the function of the molecule, thus resulting in a still active enzyme. Retention of the desired activity can be readily determined by performing standard ABTS oxidation methods, such as those described in this example.
Proteins can be used in many different industrial processes. These processes include solution polymerization of both lignin kraft and lignosulfate to produce high molecular weight lignin. Neutral / alkaline laccase is particularly advantageous in that kraft lignin is more soluble at higher pH. Such methods are described, for example, by Jin et al., Holzforshung.45 (6): 467-468,1991; U.S. Pat. No. 4,432,921; EP 0,275,544; PCT / DK93 / 00217,1992.
The laccase of the present invention can also be used for in-situ depolymerization of lignin in kraft pulp, thereby producing a pulp having a low lignin content. The use of laccase is superior to the current use of chlorine for the depolymerization of lignin. The use of chlorine leads to the production of chlorinated aromatics, which is an environmentally undesirable byproduct of the paper mill. Such use is, for example, Current opinion, Biotechnology3: 261-266,1992; J. Biotechnol.twenty five: 333-339,1992; Hiroi et al., Svensk paperstidning5: 162-166,1976. Because the environment in paper mills is generally alkaline, the laccase is more useful for this purpose than other known laccases that work best under acidic conditions.
Oxidation of dyes and dye precursors, as well as other chromogenic compounds, leads to decolorization of the compounds. Laccases can be used for this purpose, which can be very advantageous when dye transfer between fabrics is undesirable, for example in situations in the textile and detergent industries. Methods for inhibiting dye transfer and oxidation of dyes are WO92 / 01406; WO92.18683; EP 0,495,836; Calvo, Mededelingen van de Faculteit Landboum-wetenschappen / Rijiksuniversitet Gent.56: 1565-1567, 1991; Tsujino et al., J. Soc. Chem.42: 273-282,1991.
Laccase is very well suited for use in dyeing hair. In such applications, the laccase is contacted with a dye precursor, preferably on the hair, thereby achieving controlled oxidation of the dye precursor, which precursor becomes a dye or a pigment-forming compound such as a quinoid compound. Converted. The dye precursor is preferably an aromatic compound belonging to any of the three main chemical families, namely diamines, aminophenols (or aminonaphthols) and phenols. The dye precursors can be used alone or in combination. At least one of the intermediates in the copolymerization must be ortho- or para-diamine or aminophenol (primary intermediate). Examples of such are found in Chapter IV below and are p-phenylene-diamine (pPD), p-toluylene-diamine, chloro-p-phenylenediamine, p-aminophenol, o-aminophenol, 3,4- Diaminotoluene is included. U.S. Pat. No. 3,251,742 also describes other compounds, the contents of which are incorporated herein by reference. In one embodiment, the starting materials include not only enzymes and primary intermediates, but also modifiers (or couplers) (or combinations of modifiers), which are generally meta-diamines, meta-aminos. Phenol or polyphenol. Examples of modifying compounds include m-phenylene-diamine, 2,4-diaminoanisole, α-naphthol, hydroquinone, pyrocatechol, resorcinol and 4-chlororesorcinol. The modifier is then reacted with the primary intermediate in the presence of laccase to convert it to a useful compound. In another embodiment, laccase may be utilized to directly oxidize the primary intermediate to a colored compound. In all cases, the dyeing step may be performed with one or more primary intermediates, alone or in combination with one or more modifiers. Depending on the amount of the components, the usual commercial amounts for similar components, and the ratio of the components can be varied accordingly.
The use of this laccase is more traditional₂O₂This is superior to the use of the latter in that the latter causes damage to the hair and that use usually requires a high pH (which also damages the hair). In contrast, the reaction with laccase can be carried out even at alkaline, neutral or acidic pH, and the oxygen required for oxidation comes from the atmosphere rather than through harsh chemical oxidation. The result provided by the use of Myserioftra laccase is H₂O₂It is comparable to that achieved by the use of, which is not only in color development but also in washing stability and loss of brightness. A further commercial advantage resides in single container packaging in an oxygen-free atmosphere of laccase and precursor, such a method being H₂O₂The use of is impossible.
The laccase can also be used for the polymerization of phenolic compounds present in the liquid. An example of such utility would be the treatment of juice, such as apple juice, where laccase promotes the precipitation of phenolic compounds present in the juice, thus making a more stable juice produced. Such applications include Stutz and Fruit processing.7/93, 248-252, 1993; Maier et al., Dt. Lebensmittel-rindschau86 (5): 137-142,1990; Dietrich et al., Fluss.Obst57 (2): 67-73,1990.
Laccases such as Myserioftra laccase are also useful in soil detoxification (Nannipieri et al., J. Environ. Qual.20: 510-517,1991; Dec and Bollag, Arch.Environ.Contam.Toxicol.19: 543-550,1990).
The invention is further illustrated by the following non-limiting examples.
Example
I. Myseriophora thermophila laccase gene Isolation of
A. Materials and methods
1. DNA extraction and hybridization analysis
Total cellular DNA was obtained from fungal cells of Myseriophora thermophila strain E421 grown in 25 ml YEG medium (0.5% yeast extract, 2% glucose) for 24 hours using the following protocol: Extracted: Mycelium was collected by filtration through Miracloth (Calbiochem) and washed once with 25 ml TE buffer. The extra buffer was removed from the mycelium and the mycelium was frozen in liquid nitrogen. The frozen mycelium was crushed to a fine powder with an electric coffee grinder and the powder was added to 20 ml TE buffer and 5 ml 20% SDS (w / v) in a disposable plastic centrifuge tube. The mixture was gently inverted several times to ensure mixing and extracted twice with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). Sodium acetate (3M solution) was added to a final concentration of 0.3M and the nucleic acid was precipitated with 2.5 volumes of ice-cold ethanol. The tubes were centrifuged at 15,000 × g for 30 minutes and the pellet was air dried for 30 minutes and then resuspended in 0.5 ml TE buffer. DNase-free ribonuclease A was added to a concentration of 100 μg / ml, and the mixture was incubated at 37 ° C. for 30 minutes. Proteinase K (200 μg / ml) was added and each tube was further incubated at 37 ° C. for 1 hour. Finally, each sample was extracted twice with phenol: chloroform: isoamyl alcohol, and then DNA was precipitated with sodium acetate and ethanol. The DNA pellet was dried in vacuo, resuspended in TE buffer and stored at 4 ° C.
Total cellular DNA samples from transformed and untransformed control strains are analyzed by Southern hybridization. Approximately 5 μg of DNA is digested with EcoR I and size fractionated on a 1% agarose gel. The gel is photographed under short wavelength UV and soaked in 0.5 M NaOH, 1.5 M NaCl for 15 minutes and then in 1 M Tris-HCl, pH 8, 1.5 M NaCl for 15 minutes. Capillary DNA in gel in 20x SSPE (RWDavis et al., Advanced Bacterial Genetics, A Manual for Genetic Engineering. Cold Spring Habor press. 1980) on Zeta-Probe ™ hybridization membrane (BioRad Laboratories) Transfer by blotting. The membrane is baked under vacuum at 80 ° C. for 2 hours and soaked in the following hybridization buffer at 45 ° C. with gentle agitation: 5 × SSPE, 35% formamide (v / v), 0.3% SCS, 200 μg / ml denatured and sheared salmon testis DNA. A laccase-specific pro-fragment (approximately 1.5 kb) encoding the 5 'region of the N. classa lcc 1 gene was obtained from N. classa genomic DNA using standard PCR conditions (Perkin-Elmer Cetus, Emeryville, CA). Amplify using the following primer pair: forward primer 5'CGAGACTGATAACTGGCTTGG 3 '; reverse primer 5'ACGGCGCATTGTCAGGGAAGT 3'. The amplified DNA segment is first cloned into a TA-cloning vector (Invitrogen, Inc., San Diego, Calif.), Then purified by agarose gel electrophoresis and digested with EcoR I. The purified probe fragment is α [³²P] Radiolabeled by nick translation with dCTP (Amersham) and about 1 x 10 / ml buffer⁶Add to hybridization buffer at cpm activity. The mixture is incubated overnight at 45 ° C. in a shaking bath. After incubation, the membrane is washed once at 45% with 0.2 × SSPE and 0.1% SDS and then twice with 0.2 × SSPE (no SDS) at the same temperature. The membrane is dried on a paper towel for 15 minutes, then wrapped in Saran Wrap ™ and exposed to X-ray film at −70 ° C. with an intensifying screen (Kodak) overnight.
2. DNA library and identification of laccase clones
A genomic DNA library is constructed in the bacteriophage cloning vector λ-EMBL4 (J. A. Sorge. Vectors, A Snrvey of Molecular Cloning Vectors and Their Uses, Rodrigues et al., Ed pp43-60, Butterworths, Boston, 1988). Briefly, precellular DNA is partially digested with San 3A and size fractionated on a low melting point agarose gel. A DNA fragment running between 9 kb and 23 kb is excised and eluted from the gel using β-agarase (New England Biolabs, Beverly MA). The eluted DNA fragment was ligated to a BamH 1-cut and dephosphorylated λ-EMBL4 vector arm and the ligation mixture was packaged using a commercial packaging extract (Stratagene, La Jolla, Calif.) To do. The packaged DNA library is plated and propagated on Escherichia coli K802 cells. Approximately 10,000-20,000 plaques from each library are screened by plaque hybridization with radiolabeled lcc 1 DNA fragment using the conditions described above. Plaques that give a hybridization signal with this probe were purified twice on E. coli K802 cells, and DNA from the corresponding phage was amplified using Qiagen Lamda kit (Qiagen, Inc., Chatswarth, CA). Purify from titer lysate.
3. Analysis of laccase gene
Restriction mapping of laccase clones is performed using standard methods (Lewin, Genes, 2nd edition, Wiley & Sons, 1985, New York). DNA sequencing is performed with an Applied Biosystems model 373A automated DNA sequencer (Applied Biosystems. Inc., Foster City, Calif.) Using primer-walking technology with dye-terminator chemistry (H. Giesecke et al., J. Virol. Methods38: 47-60, 1992). Oligonucleotide sequencing primers are synthesized on an Applied Biosystems model 394 DNA / RNA synthesizer.
B. Results and discussion
1. Identification of laccase gene sequence
Total cellular DNA samples are prepared from Neurospora crassa, B. cinerea and Myserioftra species. Aliquots of these DNA preparations are digested with BamH 1 and fractionated by agarose gel electrophoresis. The DNA in the gel was blotted onto a Zcta-Probe ™ membrane filter (BioRad Laboratories, Hercules, Calif.), And mildly labeled with a radiolabeled fragment encoding part of the N. classa lcc 1 gene as described above. Probing under stringent conditions. Laccase-specific sequences are detected in the genomes of M. thermophila and N. crassa control but not in this probe in B. cinerea genomic DNA.
2. Myseri offtra thermophila laccase (Mt L) Gene cloning and characterization
Approximately 20,000 plaques from the M. thermophila genomic DNA library constructed in the λ-EMBL4 cloning vector are screened. This library consisted of approximately 10,000 independent clones and inserts ranged in size from 9 kb to 23 kb. Average insert size for M. thermophila is 10 kb and total genome size is 4 × 10⁷Assuming bp, this number is about 2.5 times the number of clones needed to display the entire genome. Eight plaques were identified that hybridized strongly with the N. crassa laccase gene. DNA is isolated from three of them, digested with EcoR I, and analyzed by agarose gel electrophoresis and Southern hybridization. All three of these clones hybridize with laccase specific probes. Contains a 7.5 kb EcoR I fragment. One of these EcoR I fragments is subcloned into pBR322 (Bolivar et al., Gene 2: 95-113, 1977) to create plasmid pRaMB1. A restriction map of this DNA segment is shown in FIG. The position of the laccase coding region on this clone is determined by hybridization with the lcc 1 gene fragment described above. Based on the resulting map data and the estimated size of the laccase protein of approximately 80 kdal, the entire M. thermophila laccase coding region was determined to contain a 3.2 kb Nhe I-Bgl II segment, which was identified as pUC119 (Viera and Messing , Methods Enzymol. 153: 3-11, 1987). The nucleotide sequence of this segment is determined using the primer walking method (Giesecke et al., Supra). The nucleic acid sequence is shown in FIG. 2 and SEQ ID NO: 1.
The deduced amino acid sequence of MtL can be based on amino acid sequence homology with N. crassa laccase. At the amino acid level, these two laccases share about 60% sequence identity. The similarity is highest in the region corresponding to the four histidines and one cysteine involved in the formation of the trinuclear copper cluster (Perry et al., J. Gen. Microbiol. 139: 1209-1218, 1993; Coll et al., Appl. Environ. Microbiol. 59: 4129-4135, 1993; Messerschmidt et al., J. Mol. Biol. 206: 513-530, 1989). There are 11 potential sites for N-linked glycosylation in the deduced amino acid sequence of MtL. The first 22 amino acids of MtL were found to comprise a standard signal peptide with a putative cleavage site behind the Ala residue (von Heijne, J. Mol. Biol. 173: 243-251, 1984). The amino terminus sequence of native MtL is unknown, but the amino terminus of recombinant MtL produced in A. oryza is blocked by a pyro-glutamate residue. Enzymatic removal of this residue followed by amino acid sequencing suggests that mature MtL begins with a Gln residue (position 1 in FIG. 2; SEQ ID NO: 2). Thus, it is clear that MtL is synthesized as a 620 amino acid preproenzyme having a 22 amino acid signal peptide and a 25 residue propeptide. Neurospora crassa laccase (NcL) is similarly processed at its amino terminus. In addition, NcL is proteolytically processed at its C-terminus, resulting in the removal of 13 amino acids (Germann et al., J. Biol. Chem. 263: 885-896, 1988). The processing site is the sequence Asp-Ser-Gly-Leu^*Arg₅₅₈(Where * indicates a cleavage site). Similar sequence near the C-terminus of MtL (Asp-Ser-Gly-Leu-Lys₅₆₀) And the myserioftraenzyme is C-terminal processed (Asp-Ser-Gly-Leu)^*Lys₅₆₀), Suggesting that 12 amino acids are removed.
The position of the six introns (85, 84, 102, 72, 147 and 93 nucleotides) within the lcc 1 coding region is determined by comparing the deduced amino acid sequence of MtL with that of NcL, and the common principle of intron characteristics in filamentous fungi (Gurr Et al., Gene Structure in Eukaryotic Microbes, JR Kinghorn Silk pp93-139, IRL Press, Oxford, 1987). The 1860 nucleotide coding sequence excluding introns is guanosine and cytosine rich (65.5% GtC). The codon usage pattern for this gene reflects the strong bias (89.7%) DNA base composition for codons ending with G or C.
II. Myserioftra rakka in Aspergillus Expression
A. Materials and methods
1. Bacteria and fungal host strains
Escherichia coli JM101 (Messing et al., Nucl. Acids Res. 9: 309-321, 1981) is used as a host for the construction and routine propagation of laccase expression vectors in this study. Fungal hosts for laccase expression include Aspergillus niger strains Bo-1, AB4.1 and AB1.13 (Mattern et al., Mol. Gen. Genet. 234: 332-336) and α-amylase-deficient Aspergillus oryzae strain How The uridine requirement (pyrG) mutant of B104 is included.
2. Plasmid
Plasmid pRaMB2 contains a 3.2 kb Bgl II-Nhe I fragment of M. thermophila genomic DNA encoding MtL. The vector pMWR is pUC18 (Yanisch-Perron et al., GeneThree Three: 103-119,1985) and A. oryzae TAKA-amylase promoter and pTAKA17-derived terminator element (Christensen et al., Bio / Technol.6: 1419–1422, 1988; EP 238,023). In this vector, there is a unique Swa I site at the end of the promoter element and a single Nsi I site for directed cloning of the coding sequence at the start of the terminator. Cloning vehicle pUC518 is derived by inserting a small linker containing Nsi I, Cla I, Xho I and Bgl II restriction sites between adjacent BamH 1 and Xba I sites of pUC118 (Vieira and Messing, supra). . Plasmid pToC68 (WO91 / 17243) contains the A. oryzae TAKA-amylase promoter and A. niger glaA terminator, and pToC90 (WO91 / 17243) carries the A. nidulans amdS gene.
3. Construction of laccase expression vector
The construction technique for the laccase expression vector pRaMB5 is outlined in FIG. Promoters that direct transcription of the laccase gene are obtained from the A. oryzae α-amylase (TAKA-amylase) gene (Christensen et al., Supra) and the TAKA-amylase terminator region. This plasmid is constructed by modifying pMWR3 by inserting a small rekan containing an Apa I site between the Swa I and Nsi I sites to create a plasmid called pMWR3-SAN. Pfu I polymerase dependent PCR (Stratagene, La Jolla, Calif.) Is used to amplify a short DNA segment encoding the 5 ′ portion of the MTL from the start codon to the internal Pst I site (approximately 0.5 kb). The forward primer for this PCR reaction is designed to create an EcoR I site immediately upstream of the start codon. The amplified fragment is then digested with EcoR I and Pst I (during this step, the EcoR I site is blunted (adherently terminated) by treatment with dNTP and DNA polymerase I (Klenow fragment)) and an agarose gel Purify by electrophoresis. The 3 ′ part of the M. thermophila coding region is excised from pRaMB2 as a 2 kb Pst I-Apa I fragment (this segment also contains about 110 bp from the 3 ′ untranslated region). These two fragments are combined with SwaI- and ApaI-cleaved pMWR3-SAN in a three-part ligation reaction to produce the laccase expression vector pRaMB5.
4. Transformation of Aspergillus host cells
Methods for cotransformation of Aspergillus strains are described in Christensen et al., Supra. For introduction of the laccase expression vector into A. oryza HowB104pyrG, use an equal amount (approximately 5 μg each) of the laccase expression vector and one of the following plasmids: pPYRG (Fungal Genetics Stock Center, Kansas City, KS) [this Contains the A. nidulans pyrG gene (Oakley et al., Gene61 385-399, 1987)]; pSO2 [which has the clone A. oryzae pyrG gene]; pPRYG24 [which contains the A. ficuum (= A. Niger) pyrG gene]. Primitive nutrition (Pyr⁺) Transformants were selected on Aspergillus minimal medium (Rowlands and Turner, Mol. Gen. Genet. 126: 201-216, 1973) and the transformants were selected from 1 mM 2,2′-azinobis (3-ethyl). Screen for the ability to produce laccase on minimal medium containing benzthiazoline sulfonic acid (ABTS). Cells that secrete active laccase oxidize ABTS, resulting in a green circle surrounding the colony. Finally, an equivalent amount of A. niger Bo-1 protoplast (about 5 μg each) and an A. nidulans amdS (acetamidase) gene (Hynes et al., Mol. Cell Biol.3: 1430-1439, 1983) containing pToC90. amdS⁺Transformants were selected on Cove minimal medium (Cove, Biochim. Biophys. Acta 113: 51-56, 1966) with 1% glucose as the carbon source and acetamide as the only nitrogen source, and 1 mM. Screen for laccase expression on cove medium containing ABTS.
5. Analysis of laccase-producing transformants
Transformants producing laccase active on agar plates are then purified twice through making conidia and spore suspensions in sterile 0.01% Tween-80. Optically assess the density of spores in each suspension (A₅₉₅nm). Approximately 0.5 absorption units of spores are used to inoculate 25 ml of ASPO 4 or MY50 medium in a 125 ml plastic flask. The culture is incubated at 37 ° C. with great aeration (about 200 rpm) for 4-5 days. Culture medium is obtained by centrifugation and the value of laccase activity in the supernatant is determined using syringaldazine as a substrate. Briefly, 800 μl assay buffer (25 mM sodium acetate, pH 5.5, 40 μM CnSO_Four) With 20 μl culture supernatant and 60 μl 0.28 mM syringaldazine (Sigma Chemical Co., St. Lonis, MO) in 50% ETOH. Absorption at 530 nm is measured over time with a Genesys 5 UV-bis photometer (Milton-Roy). One laccase unit (LACU) is defined as the amount of enzyme that oxidizes 1 μmole of substrate per minute at room temperature. SDS-polyacrylamide gel electrophoresis (PAGE) is performed using a precast 10-27% gradient gel from Novex (San Diego, Calif.). The protein band is developed using Coomassie Brilliant Blue (Sigma).
B. Results and discussion
1. Expression of Myserioftra laccase
Laccase producing transformants are detected by the incorporation of ABTS into the selective medium. By using pyrG or amdS as a selection marker, the co-transformation frequency varies from about 30% to 70%. The heterologous expression of MtL was found to be highest in A. oryzae transformation. Furthermore, it was found that the production rate in ASPO 4 medium was better than that in MY50, but the reason for this is not known. SDS-PAGE of the culture sample shows a band of the major laccase at approximately 80 kdal, which is similar to the size of the natural enzyme purified from M. thermophila. Similar analysis of culture filtrates from A. niger Bo transformants suggests that the laccase band is masked by very strong glucoamylase and acid stable amylase protein bands. The results are shown in Table 1.

2. Expression with or without excess copper
Aspergillus oryzae transformant HowB104-pRaMB5.30 (about 10⁹1 ml aliquots of spore suspension of aseptic spores / ml) aseptically in 100 ml sterile shake flask medium (maltose 50 g / 1; MgSO_Four・ 7H₂O 2g / 1; KH₂PO_Four 10g / 1; K₂SO_Four 2g / 1; GaCl₂・ 2H₂O 0.5 g / 1; citric acid 2 g / 1; yeast extract 10 g / 1; trace metal [ZnSO_Four・ 7H₂O 14.3g / 1; CuSO_Four・ 5H₂O 2.5g / 1; NiCl₂・ 6H₂O 0.5g / 1; FeSO_Four・ 7H₂O 13.8g / 1; MnSO_Four・ H₂O 8.5g / 1; citric acid 3.0g / 1], 0.5ml / 1; urea 2g / 1; make up with tap water and adjust to pH 6.0 before autoclaving) into a 500ml shake flask And incubate for 18 hours at 200 rpm on a rotary shaker at 37 ° C. Aseptically transfer 50 ml of this culture to 1.8 liters of fermentation medium (MgSO_Four・ 7H₂O 2g / 1; KH₂PO_Four 2g / 1; citric acid 4g / 1; K₂SO_Four 3g / 1; CaCl₂・ 2H₂Transfer to a 3 liter fermentor containing O 2g / 1; Trace metal 0.5ml / 1; Pluronic foam inhibitor 1ml / 1). The fermenter temperature is maintained at 34 ° C. by circulating cooling water through the fermenter jacket. Aseptic air is sprayed on the fermentor at a rate of 1.8 liters / minute (1 v / v / m). The agitation rate is approximately the lowest level required to keep the dissolved oxygen level in the culture above 20%. Maintain at 600-1300 rpm. Peristor aseptic additive (Nutriose 725 [maltose syrup] 225g / 1; urea 30g / 1; yeast extract 15g / 1; pluronic foam inhibitor 1.5ml / 1; make up with distilled water and autoclave) Add to fermentor using pump. The feed rate during fermentation is as follows: 30 g of additive initially before inoculation; 0-24h 2g / 1h; 24-48h 4g / 1h; 48h-end 6g / 1h.
Copper is prepared as a 400 × stock in water or a suitable buffer, sterile filtered and aseptically added to the tank to a final level of 0.5 mM. The above fermentation is also performed without adding the copper additive to the tank medium. Samples for determination of enzyme activity are withdrawn and filtered through Miracloth to remove mycelium. These samples are assayed for laccase activity by the LACU assay described above. Laccase activity is observed to increase continuously during the fermentation, and a value of about 45 LACU / ml is achieved after 180 hours in fermentation with excess copper. At a specific activity of 22 LACU / mg, this corresponds to 2 g / 1 expressed recombinant laccase. On the other hand, the maximum laccase activity achieved in the fermentation without copper additive was about 10 LACU / ml after 170 hours, about 25% of that found in the presence of added copper.
III. Purification and characterization of Myserioftra laccase
A. Materials and methods
1.Material
The chemical reagents used as buffers and substrates should be at least reagent grade products. Endo / N-glycosidase F and pyroglutamate aminopeptidase were purchased from Boehringer Mannheim. Chromatography is performed either on a Pharmacia FPLC or a conventional low pressure system. Absorbance assays are performed with either a photometer (Shimadzu PC160) or a microplate reader (Molecular Devices). Britton & Robinson (B & R) buffer is prepared according to the protocol described in Quelle, Biochemisches Taschenbuch, H. M. Raven, II. Teil, S. 93u, 102, 1964.
2.Enzyme activity
Laccase activity is determined by syringaldazine oxidation in a 1-cm quartz cuvette at 30 ° C. Mix 60 μl of syringaldazine stock solution (0.28 mM in 50% ethanol) and 20 μl of sample with 0.8 ml of preheated buffer solution. Oxidation is monitored at 530 nm for 5 minutes. Activity is expressed as μmole of substrate oxidized per minute. Use various pH B & R buffers. The active unit is referred to herein as “SOU”. 25 mM sodium acetate, 40 μM CuSO to determine the activity referred to as LACU as described above_FourAlso use a pH 5.5 buffer. 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) oxidation assay uses 0.4 mM ABTS, B & R buffer, pH 4.1, and A at room temperature.₄₀₅This is done by monitoring. ABTS oxidase activity upper layer (overlay) assay consists of chilled ABTS-agarose (0.05 g ABTS, 1 g agarose, 50 ml H₂O, heated to dissolve the agarose) is poured onto a natural IEF gel and incubated at room temperature. Thermal stability analysis of laccase (r-MtL) is performed using samples with 3SOU activity pre-incubated at various temperatures in B & R buffer pH6. Samples are diluted 400-fold in the same buffer and then assayed at room temperature.
3. Purification from fermentation broth
3.7 liters of cheesecloth filtered culture (pH 7.6, 16 mS) is filtered through Whatman # 2 filter paper. The culture solution is concentrated from 3700 ml to 200 ml using a spiral concentrator (Amicon) with an S1Y100 membrane (MWCO: 100). The concentrate is adjusted to 0.75 mS by diluting in water and reconcentrated to 170 ml with S1Y100. The washed and concentrated culture has a dark green color.
The culture is frozen overnight at −20 ° C., thawed the next day, and loaded onto a Q-sepharose XK26 column (120 ml) that has been pre-equilibrated with 10 mM Tris, pH 7.5, 0.7 mS (buffer A). The blue laccase band slowly descends the column during the addition. One group of blue fractions passes through the column after addition and washing with buffer A. The second group elutes during a linear gradient with buffer B (buffer A and 2M NaCl). Some brown material without laccase activity is subsequently eluted with 1M NaOH. SDS-PAGE analysis shows that this preparation yields pure laccase.
4. Amino acid content, degree of glycosylation and N-terminal configuration Analyzing columns
N-terminal sequencing is performed on an ABI 476A sequencer. Total amino acid analysis (thereby determining the r-MtL excitation coefficient) is performed on an HP Amino Quant instrument. Deglycosylation is performed using endo / N-curcosidase F according to manufacturer's specifications and carbohydrate content is assessed by the difference in mobility as determined by SDS-PAGE. N-terminal deblocking with pyroglutamate aminopeptidase is performed according to manufacturer's specifications. Approximately 80 μg of r-MtL is treated with 4 μg of peptidase in the presence or absence of 1 M urea or 0.1 M guanidine HCl and then blotted onto a PVDF membrane for sequencing. About 20 pmol of deblocked protein is obtained and sequenced.
SDS-PAGE and natural IFF analysis are performed in either Novex cells or Mini Protean II and Model III Mini IEF cells (Bio-Rad). Gel filtration analysis was performed with Sephacryl S-300 (Pharmacia), from which blue dextran (2000 kdal), bovine IgG (158 kdal), bovine serum albumin (66 kdal), ovalbumin (45 kdal) and horse to calibrate the column. Estimated by using cardiac myoglobin (17 kdal).
B. Results and discussion
1. Purification and characterization of r-MtL from fermentation broth
About 2-3 g of r-MtL is isolated from 3.71 fermentation broth. Initial concentration using a membrane with 100 kdal MWCO removed a significant amount of brown material and a small amount of contaminating protein. The low affinity of r-MtL for Q-Sepharose matrix equilibrated with 10 mM Tris, pH 7.5 facilitates its separation from other more acidic and more strongly bound impurities. As shown by SDS-PAGE, this preparation resulted in essentially pure laccase for the most active fraction located around the peak. Other weakly active fractions can be further purified on either a Mon-Q or gel filtration column with a shallower gradient, such as S-300, from which contaminants are separated based on the smaller MW. Overall, 18 times purification and 67% recovery can be achieved. As described below, the presence of two elution bands of r-MtL in Q-Sepharose chromatography is probably based on differential glycosylation.
The purified r-MtL shows 100-140 kdal MW on S-300 gel filtration and 85 kdal MW on SDS-PAGE. An increase in r-MtL mobility on SDS-PAGE after deglycosylation indicates that carbohydrate accounts for 14% of its total mass. Natural IEF showed a major band of pi-4.2 active in the ABTS overlay assay.
Direct sequencing of the N-terminus of purified r-MtL from either desalted solution or PVDF membrane samples was unsuccessful. However, treatment of r-MtL with pyroglutamate aminopeptidase resulted in a protein with a deblocked N-terminus. This is not observed in the processing of propeptides during r-MtL maturation, ie other laccases such as Rhizoctonia solani, but a post-translational phenomenon similar to that of N. crassa laccase To suggest. Possible schemes are outlined below.

The blue r-MtL spectrum had maximum absorption at 276 and 589 nm.
The activity of laccase is tested using either syringaldazine or ABTS as a substrate. Abs₂₇₆Expressed as per or per mg, the laccase had a value of 20 or 45 units for SOU, respectively, at pH 6.5. LACU assay is Abs₂₇₆This gave a value of 10 or 22 units per or mg.
The pH profile of r-MtL activity is quite close to that of wild type, with an optimum pH of 6.5. The temperature value for full activity retained after the 20 minute pre-incubation observed for r-MtL is about 60 ° C. Purified r-MtL showed no activity loss after freezing at -20 ° C in Q-Sepharose elution buffer and storing for 5 weeks.
When comparing the two forms of r-MtL obtained from fermentation broth isolated with Q-Sepharose, SDS-PAGE, natural PAGE, natural IEF, S-300 gel filtration, UV-visible spectrum, syringaldazine and There was no significant difference in terms of specific activity against ABTS, as well as deblocked N-terminal sequencing scale. Similarly, the different elution patterns on Q-Sepaharose are derived from several different glycosylation.
IV. Myseriophora laccase in hair dyeing Use of
The dyeing effect of Myserioftra laccase was tested on the basis of various dye precursors and also on 0.1% p-phenylenediamine contrasted with some modifiers.
material:
Dye precursor:
0.1% p-phenylene-diamine in 0.1M K-phosphate buffer (pH 7.0)
0.1% aminophenol in 0.1M K-phosphate buffer (pH 7.0)
enzyme:
Recombinant Myserioftra thermophila laccase 16LACU / ml (in final staining solution).
apparatus:
Datacolor Textflash 2000 (CIE-Lab)
Evaluation of hair color
The qualitative color of curly hair is Datacolor Textflash 2000, CIE-Lab parameter L^*("0" = black and "100" = white) and a^*(“−” = Green, and “+” = red).
result:
Dyeing effect
European blond curly hair (1 g) is used to test Myseri offtra thermophila laccase for oxidative hair dyeing. P-Phenylenediamine and o-aminophenol are used as dye precursors.
Hair dyeing
4 ml of dye precursor solution is mixed with 1 ml of laccase in a Whirley mixer, applied to curly hair and kept at 30 ° C. for 60 minutes. Rinse the curly hair with tap water approximately 3 times, hold it between two fingers, pass the comb, and air dry.
The results of the dyeing effect test are shown in Tables 1 and 2 below.

Test results
From Tables 1 and 2, it can be seen that Myserioftra thermophila laccase can be used for oxidative dyeing of hair.
Deposit of biological materials
The following biological materials were deposited under the Budapest Treaty on May 25, 1994 at the Agricultural Research Service Patent Culture Collection, Other Regional Research Center, 1815 University Street, Peoria, Illinois, 61604, and the following accession numbers: Is given.
Deposit              Accession number
E. coli JM101 NRRL B-21261 containing pRaMB5
Sequence listing
(1) General information:
(I) Applicant:
(A) Name: Novo Nordisk Biotech, Inc.
(B) Street: 1445 Drew Avenue
(C) City: Davis, California
(D) Country: United States of America
(E) Zip code: 95616-4880
(F) Phone number: (916) 757-8100
(G) Fax number: (916) 758-0317
(I) Applicant:
(A) Name: Novo Nordisk A / S
(B) Street: Novo Alle
(C) City: Bagsv rd
(D) Country: Denmark
(E) Zip code: DK-2880
(F) Phone number: +45 4444 8888
(G) Fax number: +45 4449 3256
(Ii) Title of invention: Purified Myserioftra laccase and nucleic acid encoding the same
(Iii) Number of sequences: 2
(Iv) Contact information:
(A) Name: Novo Nordisk of North America, Inc.
(B) Street: 405 Lexington Avenue, Suite 6400
(C) City and state: New York, New York
(D) Country: U.S.A.
(E) Zip code: 10174-6401
(V) Computer reading form:
(A) Media type: Floppy disk
(B) Computer: IBM PC compatible
(C) Operating system: PC-DOS / MS-DOS
(D) Software: PatentIn Release # 1.0, Version # 1.25 (EPO)
(Vi) Current applicant data:
(A) Application number: Before certification
(B) Application date: May 31, 1995
(C) Classification:
(Vii) Previous application data:
(A) Application number: US 08 / 253,781
(B) Application date: June 3, 1994
(Viii) Agent / Agent Information:
(A) Name: Lowney, Karen A.
(B) Registration number: 31,274
(C) Reference / case number: 4184.204-WO
(Ix) Communication information:
(A) Phone number: 212 867 0123
(B) Fax number: 212 867 0298
(2) Information about SEQ ID NO: 1:
(I) Sequence features:
(A) Length: 3187 base pairs
(B) Type: Nucleic acid
(C) Number of strands: double strand
(D) Topology: straight chain
(Ii) Molecular type: DNA (genome)
(Vi) Origin:
(A) Organism: Myseri offtra thermophila
(Ix) Features:
(A) Name / Key: Intron
(B) Location: 833 ... 917
(Ix) Features:
(A) Name / Key: Intron
(B) Location: 996 ... 1077
(Ix) Features:
(A) Name / Key: Intron
(B) Position: 1090 ... 1188
(Ix) Features:
(A) Name / Key: Intron
(B) Position: 1261 ... 1332
(Ix) Features:
(A) Name / Key: Intron
(B) Position: 2305 ... 2451
(Ix) Features:
(A) Name / Key: Intron
(B) Position: 2521 ... 2613
(Ix) Features:
(A) Name / Key: CDS
(B) Location: Communication (587..832,918..995,1078..1089,1189..1260,1333..2304,2452..2520,2614..3024)
(Xi) Sequence details: SEQ ID NO: 1:

(2) Information about SEQ ID NO: 2:
(I) Sequence features:
(A) Length: 620 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: straight chain
(Ii) Molecular type: protein
(Vi) Origin:
(A) Organism: Myseri offtra thermophila
(Xi) Sequence details: SEQ ID NO: 2:

Claims (42)

Myserioftra laccase comprising an amino acid sequence that is 90% or more homologous to the amino acid sequence described in SEQ ID NO.2. Myserioftra laccase comprising an amino acid sequence that is 95% or more homologous to the amino acid sequence described in SEQ ID NO.2. The laccase according to claim 1, which is Myserioftra thermophila laccase. Ru comprising the amino acid sequence of SEQ ID NO.2, Myceliophthora, laccase according to claim 1, wherein. The laccase according to claim 1, having a specific activity of 30 SOU / mg or more with respect to syringaldazin at an optimum pH. A DNA construct comprising a nucleic acid encoding the myserioftra laccase according to claim 1. A DNA construct comprising a nucleic acid encoding the myserioftra laccase according to claim 2. A DNA construct comprising a nucleic acid encoding the myserioftra laccase according to claim 3. A DNA construct comprising a nucleic acid encoding the myserioftra laccase according to claim 4. A DNA construct comprising a nucleic acid encoding the myserioftra laccase according to claim 5. A recombinant vector comprising the DNA construct according to claim 6. 12. The vector of claim 11, wherein the construct is operably linked to a promoter sequence. 13. The vector according to claim 12, wherein the promoter is a fungal or yeast promoter. 14. The vector of claim 13, wherein the promoter is an Aspergillus oryzae TAKA amylase promoter. 14. The vector of claim 13, wherein the promoter is an Aspergillus niger or Aspergillus awamori glucoamylase (gluA) promoter. 12. A vector according to claim 11, which also comprises a selectable marker. 17. The vector of claim 16, wherein the selectable marker is selected from the group consisting of amds, pyrG, argB, niaD, sC and hyg B. The vector according to claim 16, wherein the selection marker is an amdS marker of Aspergillus nidulans or Aspergillus oryza, or a pyrG marker of Aspergillus nidulans, Aspergillus niger, Aspergillus awamori or Aspergillus oryzae. And TAKA amylase promoters Aspergillus oryzae, comprising both the amdS or pyrG marker of Aspergillus nidulans or Aspergillus oryzae, vector of claim 16, wherein. A recombinant host cell comprising the heterologous DNA construct of claim 6. 21. The cell according to claim 20, which is a fungal cell. 21. The cell according to claim 20, which is an Aspergillus cell. 21. The cell of claim 20, wherein the construct is integrated into the genome of the host cell. 21. The cell of claim 20, wherein the construct is contained on a vector. It said nucleic acid encodes a laccase having the amino acid sequence shown in SEQ ID NO.2, claim 20 of the cell. The recombinant host cells comprising a laccase according to claim 6 or 7, wherein comprising a DNA construct comprising a nucleic acid encoding cultured under conditions that induce expression of the laccase, then the enzyme from the culture A process for obtaining laccase according to any one of claims 1 to 5 , comprising recovering. Culturing a recombinant host cell comprising a DNA construct comprising a sequence encoding a copper-containing enzyme in the presence of 0.05 to 0.5 mM copper under conditions that induce expression of the enzyme. A method for increasing the yield of active recombinant laccase according to any one of claims 1-5 . 6. A method for solution polymerization of a lignin or lignosulfate substrate comprising contacting the substrate with a laccase according to any one of claims 1-5. A method for in situ depolymerization in Kraft pulp, the method comprising contacting said pulp with laccase of claims 1 to 5 gall Zureka preceding claim. A method for oxidizing a dye or dye precursor comprising contacting said dye with a laccase according to any one of claims 1-5 . A method for dyeing hair, the laccase according to any one of 請 Motomeko 1-5, the presence of at least one modifying agent or in the absence, and at least one dye precursor, Contacting the dye precursor for a time and under conditions sufficient to oxidize the dye precursor. 32. The method of claim 31, wherein the dye precursor is selected from the group consisting of diamine, aminophenol and phenol. 32. The method of claim 31, wherein the modifier, if used, is meta-diamine, meta-aminophenol or polyphenol. 33. The method of claim 32, wherein the dye precursor is a primary intermediate selected from the group consisting of ortho- or para-diamine or aminophenol. 32. The method of claim 31, wherein more than one dye precursor is used. 32. The method of claim 31, wherein more than one modifier is used. 32. The method of claim 31, wherein both a primary intermediate and a modifier are used. 6. A dye composition comprising Myserioftra laccase according to any one of claims 1 to 5, in combination with at least one dye precursor. 40. The dye composition of claim 38, further comprising at least one primary intermediate and at least one modifier. 39. A container comprising the dye composition according to claim 38 in an oxygen-free atmosphere. 41. The container of claim 40, further comprising at least one primary intermediate dye precursor combined with at least one modifier. A method for polymerizing or oxidizing a phenolic or aniline compound comprising contacting the phenolic or aniline compound with the myserioftra laccase according to any one of claims 1-5 .

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